Requirement of Calmodulin Binding by HIV-1 gp160 for Enhanced
FAS-mediated Apoptosis *
Keith J.
Micoli ,
George
Pan ,
Yong
Wu ,
John P.
Williams ,
William J.
Cook , and
Jay M.
McDonald § ¶
From the Department of Pathology, University of
Alabama at Birmingham, Birmingham, Alabama 35294and the
§ Veterans Administration Medical Center,
Birmingham, Alabama 35233
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Accelerated apoptosis is one mechanism proposed
for the loss of CD4+ T-lymphocytes in human immunodeficiency virus type
1 (HIV-1 ) infection. The HIV-1 envelope glycoprotein, gp160, contains two C-terminal calmodulin-binding domains. Expression of
gp160 in Jurkat T-cells results in increased sensitivity to FAS-
and ceramide-mediated apoptosis. The pro-apoptotic effect of gp160 expression is blocked by two calmodulin antagonists, tamoxifen and trifluoperazine. This enhanced apoptosis in response to FAS antibody or C 2 -ceramide is associated with activation
of caspase 3,a critical mediator of apoptosis. A point mutation in the
C-terminal calmodulin-binding domain of gp160 (alanine 835to
tryptophan, A835W) eliminates gp160-dependent enhanced
FAS-mediated apoptosis in transiently transfected cells, as well as
in vitro calmodulin binding to a peptide corresponding to
the C-terminal calmodulin-binding domain of gp160. Stable Tet-off
Jurkat cell lines were developed that inducibly express wild type gp160
or gp160A835W. Increasing expression of wild type gp160, but not
gp160A835W, correlates with increased calmodulin levels, increased
apoptosis, and caspase 3activation in response to anti-FAS treatment.
The data indicate that gp160-enhanced apoptosis is dependent upon
calmodulin up-regulation, involves the activation of caspase 3,and
requires calmodulin binding to the C-terminal binding domain of gp160.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
HIV-1 1 infection is
characterized by immune system hyperactivation and dysfunction that
increases with disease progression until immune function is lost.
During the long asymptomatic phase, a state of equilibrium appears
to be achieved, consisting of a rapid turnover of lymphocytes
concomitant with rapid viral production and clearance ( 1-3 ). This
equilibrium eventually shifts with a loss of the ability of the immune
system to replace CD4+ cells and a gradual increase in viral load.
Since HIV-1 primarily infects CD4+ T-lymphocytes, and the decline in
these cells along with the rise in viral load is a hallmark of disease
progression, understanding the relationship between these phenomena is
critical in understanding HIV-1 pathogenesis.
One proposed mechanism for the accelerated loss of CD4+ cells is an
increased rate of apoptosis ( 4 ). Apoptosis, or programmed cell death,
differs from necrosis in that dying cells participate actively in their
own death and generally do not induce an inflammatory response ( 5 ).
Physiologically, apoptosis functions in maintaining tissue homeostasis
and is important in removal of lymphocytes after an immune response.
The latter function prevents accumulation of lymphocytes in the blood
and is mediated by interaction of the apoptotic receptor, FAS, with FAS
ligand. Upon binding FAS ligand, FAS recruits signaling molecules to
the death-inducing signaling complex ( 6-8 ). Induction of this pathway
leads to activation of caspase 3,subsequent activation of caspase 6,and eventually activation of DNA fragmentation factor and a
Ca 2+ /Mg 2+ -dependent endonuclease
responsible for cleavage of DNA resulting in the typical laddering
pattern seen in most forms of apoptosis ( 9-11 ).
The importance of apoptosis in AIDS is controversial although there is
abundant in vitro evidence supporting a role for apoptosis in the pathogenesis of HIV-1 ( 12 ), and although mechanisms regulating this apoptosis in vivo are not clear, current evidence
implicates the FAS pathway. Evidence supporting this hypothesis
includes reports that lymphocytes from HIV-1-infected individuals
express elevated levels of FAS and are more sensitive to FAS-mediated apoptosis in vitro compared with non-infected individuals
( 13-15 ). The chronically activated immune state in HIV infection may
lead to an improperly elevated rate of apoptosis in vivo.
Additionally, the level of spontaneous apoptosis in lymphocytes from
HIV+ individuals is elevated and positively correlates with increasing
disease severity ( 12, 16 ).
The coat glycoprotein of HIV-1, gp160, is post-translationally cleaved
to an extracellular subunit, gp120, and a transmembrane subunit, gp41.
The subunits are non-covalently associated, and both are required for
viral entry into the cell. Two calmodulin-binding sites have been
identified near the C terminus of gp160 ( 17 ). Purified gp160 or
peptides corresponding to the gp160 calmodulin-binding sites bind
calmodulin in vitro and inhibit
calmodulin-dependent enzymes ( 18 ). Expression of gp160 in
Molt4 cells, a FAS-resistant human T-cell line, rendered these cells
sensitive to FAS-induced apoptosis ( 19 ), whereas this effect was
diminished in Molt4 cells expressing gp160 147, a truncated
glycoprotein that lacks both calmodulin-binding domains ( 19 ). Further
evidence implicating calmodulin in the cytopathic effect of gp160 is
that two calmodulin antagonists, tamoxifen and trifluoperazine, block
FAS-mediated apoptosis in gp160-expressing cells ( 19 ). In addition,
tamoxifen and trifluoperazine have been reported to reduce spontaneous
apoptosis in cultured peripheral blood mononuclear cells from
HIV-1-infected individuals ( 16 ).
We demonstrate here that gp160 expression enhances FAS-mediated
apoptosis in Jurkat cells by increasing calmodulin expression and
accelerating caspase 3activation and that these effects that require
calmodulin binding to gp160 are blocked by a single point mutation in
the C-terminal calmodulin-binding domain.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Cells and Cell Culture--
Jurkat cells were purchased from the
ATCC (Mannasas, VA) and grown at 37°C in 5% CO 2 in
RPMI 1640medium supplemented with 10% heat-inactivated fetal bovine
serum, 100units/ml penicillin, 100µg/ml streptomycin, and 2m M L -glutamine.
Plasmids--
Plasmid pSRHS containing the HIV-1 envelope gene,
gp160, and the truncated forms of gp160, 147, which lacks the
C-terminal 147amino acids of gp41, and 67, which lacks the
C-terminal 67 amino acids of gp41, as well as the plasmid pKS8,
containing the cDNA for -galactosidase under control of the
human -actin promoter, were kindly provided by Eric Hunter,
Department of Microbiology, University of Alabama at Birmingham, and
were used in all transfection experiments.
Transfections--
All transient transfections were performed
using the cationic lipid, DMRIE-C (Life Technologies, Inc.). Briefly,
lipid-DNA complexes were allowed to form for 45min at room temperature in serum-free medium. Cells were added to the complex in serum-free medium and incubated for 5h at 37°C in 5% CO 2.
RPMI 1640medium (Life Technologies, Inc.) was added, and cells were
cultured an additional 48h. Transfection efficiency is monitored
by simultaneous transfection of the -galactosidase expression
vector, pKS8. Efficiencies ranged from 50to 90% in Jurkat cells.
Antibodies and Reagents--
Monoclonal antibody to calmodulin
was developed as described previously ( 20 ) and is available from
Upstate Biotechnology Inc. Lake Placid, NY. Mouse anti-human FAS
monoclonal antibody (Upstate Biotechnology Inc.) was used to induce
apoptosis. Mouse anti-human caspase 3monoclonal antibody was from
Transduction Laboratories (San Diego, CA), caspase 3polyclonal
antibody was from PharMingen (San Diego, CA); C 2 -ceramide
and C 2 -dihydroceramide was from Avanti Polar Lipids
(Birmingham, AL).
Site-directed Mutagenesis--
Stratagene's (La Jolla, CA)
Quikchange Site-directed Mutagenesis Kit was used to make point
mutations of gp160 according to manufacturer's instructions. Primers
for the mutagenesis were purchased from Life Technologies, Inc., and
correspond to the desired mutation of alanine 835to tryptophan flanked
by 10-15 base pairs of correct sequence, using the sequence of HIV-1
strain HXB2 as a template.
Creation of gp160- and gp160A835W-expressing Cell
Lines--
Tet-Off Jurkat cells (, Palo
Alto, CA) were transfected with the pTRE expression vector containing
gp160 or gp160A835W cDNA by electroporation. Selection of stable
cell lines was initiated 48h after transfection using 100µg/ml
geneticin, 300µg/ml hygromycin, and 2µg/ml tetracycline in RPMI
1640complete medium changed every 4days. After 5-7 days, living
cells were separated from dead cells and plated at a lower density.
After serial dilution, isolated single cell clones were cultured in
96-well plates and then transferred into 12-well and 6-well plates and
a 25-cm 2 T flask.
Northern Blot of gp160 and gp160A835W in Tet-off Jurkat Clones
Induced by Removal of Tetracycline--
Jurkat clones expressing HIV-1
gp160 and mutant gp160A835W were incubated with decreasing doses of
tetracycline for 48h. Total RNA was isolated by the guanidinium
thiocyanate/phenol/chloroform method and separated by formaldehyde gel
electrophoresis. RNA was blotted onto Hybond N membranes and
prehybridized for 45min at 68°C and then hybridized for 1h
using Quikhyb (Stratagene). The hybridization probe was generated by
random labeling with Prime-a-Gene Labeling System (Promega, Madison,
WI) with [ - 32 P]dCTP and HIV-1 HXB2 envelope cDNA template.
Peptide Synthesis--
Peptides corresponding to the C-terminal
calmodulin-binding site of gp160 (residues 826-843) were
synthesized at the University of Alabama at Birmingham Comprehensive
Cancer Center Peptide Synthesis and Analysis Shared Facility and
purified by high pressure liquid chromatography. Wild type gp160
peptide sequence is 826-DRVIEVVQG A CRAIRHIP-843, and
gp160A835W sequence is 826-DRVIEVVQG W CRAIRHIP-843, with the residue corresponding to 835 underlined.
TdT Apoptosis Staining--
In situ apoptosis
staining was performed using terminal deoxynucleotide transferase
(Roche Molecular Biochemicals). Briefly, cells were collected after
treatments and cytospun onto microscope slides and fixed in 10%
formalin in PBS. Slides were then rinsed and treated with 20µg/ml
Proteinase K (Fisher) for 15min at room temperature. Slides were
washed several times with water and incubated with at 37°C TdT (0.3units/ml), digoxigenin-labeled dUTP, and buffer (30m M Tris
base, pH 7.2,140m M sodium cacodylate, 1m M
cobalt chloride). After a brief wash, slides were blocked in 1% bovine
serum albumin, 0.1% gelatin in PBS for 15min at room temperature.
Slides were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody for 1h at room temperature, washed in
PBS, and developed with nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Sigma) at room
temperature for 30min. Slides were coverslipped and at least 400cells
per slide were counted to determine the percentage of apoptotic cells.
Western Blot for Caspase 3and Calmodulin--
Jurkat cells were
treated as indicated and lysed in buffer containing 50m M
HEPES, pH 7.4,150m M NaCl, 10% glycerol, 1% Triton X-100, 1m M sodium orthovanadate, 10m M EDTA,
10m M EGTA, 1m M ammonium molybdate, 50m M NaF, 0.5µ M okadaic acid, 5m M
benzamidine, and 50µg/ml pepstatin. Equivalent amounts of protein
(100µg) were separated on 12.5% SDS-PAGE and transferred to
Immobilon P membrane (Millipore, Bedford, MA). Membranes were fixed in
0.2% glutaraldehyde in Tris-buffered saline (TBS ) for calmodulin only, blocked in TBS containing 2% bovine serum albumin and 0.2% gelatin, and then incubated with a monoclonal caspase 3antibody diluted 1:1000,
a polyclonal caspase 3antibody (1:3000), or a monoclonal calmodulin
antibody (1:3000) in TBS/Tween 20(TTBS) and anti-mouse or anti-rabbit
horseradish peroxidase-conjugated antibody (Amersham Pharmacia
Biotech), 1:5000 in TTBS followed by development with enhanced
chemiluminescence (Amersham Pharmacia Biotech).
Dansyl-Calmodulin Binding--
The binding of calmodulin to
peptides corresponding to the C-terminal calmodulin-binding domain of
gp160 or the A835W mutant was determined fluorimetrically with
dansyl-calmodulin (Ocean Biologics, Corvalis, OR) as reported
previously ( 21 ). Briefly, fluorescence emission of dansyl-calmodulin
was scanned from 400 to 600nm after excitation at 340nm with the
indicated concentrations of peptide. All assays were performed in
buffer containing 50 m M MOPS, pH 7.3,200m M
KCl, and 1m M CaCl 2 with or without 5 m M EGTA. Emission spectra were obtained with concentrations
of peptides ranging from 0to 1µ M, and mellitin was used
as a positive control for binding.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Enhancement of Ceramide- and FAS-mediated Apoptosis by
gp160--
Jurkat cells were transfected with gp160 or
mock-transfected with empty vector and treated with a monoclonal FAS
antibody (clone CH11), and apoptosis was determined as described under "Experimental Procedures." Similar levels of apoptosis (11% in mock-transfected and 13% in gp160 transfected) were observed in cells
not treated with FAS antibody (Fig.
A ). Cells transfected with
gp160 undergo significantly more apoptosis (35±3%) than vector-transfected cells (25±1%) after treatment with FAS
antibody ( n =8, p <0.01). There was
no difference in basal or FAS-mediated apoptosis between
vector-transfected cells and untransfected Jurkat cells (data not
shown).
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Fig. 1.
Apoptosis in control Jurkat cells and
gp160-transfected Jurkat cells treated with anti-FAS or
C 2 -ceramide. Jurkat cells were transfected using
DMRIE-C with empty vector, pSRHS, or pSHRSgp160. Forty-eight hours
post-transfection, cells were treated with 500ng/ml anti-FAS antibody
( A ) or 50µ M C 2 -ceramide or
dihydroceramide ( B ) for 3h. Cells were then collected
for TdT staining and counting. Data shown are the mean percentage
apoptotic cells of eight separate experiments, ±S.E. FAS-mediated
apoptosis in gp160-transfected cells was significantly higher than in
vector-transfected cells ( p <0.01) as was
ceramide-mediated apoptosis ( p <0.001).
Previous studies have shown that ceramide levels increase in
HIV-1-infected cells in vitro ( 22 ) and that treatment of
latently infected cells with ceramide induces viral production ( 23 ). Furthermore, ceramide is a reported second messenger in FAS-mediated apoptosis. To delineate the site of action of gp160 further, we tested
the effect of gp160 on ceramide-mediated apoptosis in Jurkat cells. The
specificity of ceramide action was demonstrated using dihydroceramide
as a negative control (Fig. B ). Dihydroceramide is a
biologically inactive molecule, which differs from ceramide by one
double bond ( 24 ). The ability of gp160 to enhance ceramide-mediated apoptosis is virtually identical to the effect of gp160 on FAS-mediated apoptosis (Fig. A ). Ceramide treatment induced
apoptosis in 32 ±3% of gp160-transfected cells compared with
22±3% in vector-transfected cells ( n =8, p <0.001). Basal levels of apoptosis (13±2% in gp160-transfected cells and 11±2% in vector alone
transfected cells) did not change in response to dihydroceramide.
Effect of Calmodulin Antagonists on gp160-enhanced Ceramide-
and FAS-mediated Apoptosis--
Jurkat cells were transfected with
gp160 as described above and pretreated with 10µ M
tamoxifen (TMX ) or trifluoperazine (TFP ) for 30min prior to addition
of FAS antibody (Fig. ). Although TMX is
widely used as an anti-estrogen, it is as potent a calmodulin antagonist as TFP in the range of 1-10 µ M ( 25 ). TMX
( n =3) and TFP ( n =5) inhibited the
FAS-mediated apoptosis in gp160-transfected cells by 75±10and
90±2%, respectively (Fig. A ).
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Fig. 2.
Inhibition of gp160 enhancement of FAS- and
ceramide-mediated apoptosis by calmodulin antagonists in Jurkat
cells. Jurkat cells were transfected with pSHRSgp160 and
pretreated with 10µ M TMX of TFP for 30min before
addition of 500ng/ml FAS antibody ( A ) or 50µ M C 2 -ceramide ( B ). Cells were
then TdT-stained and apoptosis was quantified by counting at least
400cells/slide. Percent inhibition of apoptosis was calculated by
setting the amount of apoptosis in the absence of inhibitor as 100and
apoptosis in the presence of the calmodulin antagonist as percent of
this number±S.E. Data shown represent the mean±S.E. of
three ( TMX ) or five ( TFP ) separate
experiments.
The effects of TMX and TFP on gp160 enhanced ceramide-induced apoptosis
were also tested to determine whether gp160-enhanced ceramide-mediated
apoptosis is affected by calmodulin antagonists in a way that is
similar to FAS-mediated apoptosis. Both TMX ( n =3) and
TFP ( n =8) inhibited gp160-enhanced ceramide-mediated apoptosis by 65±10and 90±5%, respectively (Fig.
B ). The data clearly indicate that the percent inhibition
observed in these experiments is similar to the effect of TFP and TMX
on FAS-mediated apoptosis in gp160-transfected cells.
Effect of gp160 Expression on Caspase 3Activation by FAS and
Ceramide--
Since gp160 enhanced both FAS- and ceramide-mediated
apoptosis, we investigated potential downstream mechanisms regulating this effect. Caspase 3is recognized as an important effector molecule
in the apoptotic pathway, and we hypothesized that gp160 expression may
alter the activity of this protease. Caspase 3, like all known
caspases, is synthesized as a zymogen and undergoes proteolytic
activation in two steps. Cleavage by an upstream caspase followed by a
second autocatalytic cleavage forms the fully active dimer of
approximately 17and 12kDa. Enzyme activation in these studies is
monitored by Western analysis for the 32-kDa proform (CPP32), which
decreases upon enzyme activation. Cells treated with either FAS
antibody or C 2 -ceramide for the indicated times demonstrate
that the rate of caspase 3activation is increased in cells transfected
with gp160 as determined by Western blot analysis of Jurkat cell
lysates from cells transfected with empty vector or vector containing
gp160 (Fig. ). The accelerated rate of
decrease in the amount of the 32-kDa proform of the enzyme in response
to both FAS antibody (Fig. A ) and C 2 -ceramide
(Fig. B ) is evident. Thus, gp160 enhances both FAS- and
ceramide-mediated apoptosis by a common mechanism involving activation
of caspase 3.
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Fig. 3.
Western blot for caspase 3from Jurkat cells
treated with anti-FAS or C 2 -ceramide. Jurkat cells
were transfected with empty vector or gp160 and treated with anti-FAS
antibody ( A ) or C 2 -ceramide ( B ) for
the times indicated. After lysis, equivalent amounts of protein were
subjected to SDS-PAGE and transferred to Immobilon P membrane and
immunoblotted for caspase 3using a monoclonal antibody to caspase 3.Shown are representative examples from at least two independent
experiments for each treatment with the inactive proform of caspase 3,CPP32, labeled.
Determination of the Amino Acid Residues of gp160 Critical to
Calmodulin Binding and Enhanced Apoptosis--
To test further the
hypothesis that gp160-enhanced apoptosis requires calmodulin binding,
we computer-modeled the three-dimensional structure of calmodulin bound
to a peptide corresponding to the native C-terminal calmodulin-binding
domain. The C-terminal rather than the N-terminal calmodulin-binding
domain was selected because FAS-mediated apoptosis was not enhanced in
cells transfected with gp160 67 (a deletion of the C-terminal
calmodulin-binding domain) compared with cells transfected with wild
type gp160 (data not shown). Previous studies with this mutant
determined that it also eliminated viral production in vitro
( 26 ). Computer modeling aligning the calmodulin-binding domain of
myosin light chain kinase (MLCK ) with the C terminus of gp160
identified five amino acids in the C-terminal calmodulin-binding domain
of gp160 likely to be important for calmodulin binding (Fig.
A, in bold ).
Calcium-bound calmodulin (Fig. B ) has two globular domains,
each containing two calcium-binding sites, separated by a highly
flexible amphipathic helical region. It is this helical region that
undergoes a major conformational change when calmodulin binds to target
proteins by wrapping around the amphipathic helix of the target protein and bringing the two globular domains into close approximation.
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Fig. 4.
Comparison of the calmodulin-binding domain
of smooth muscle myosin light chain kinase and the C-terminal
calmodulin-binding domain of HIV-1 . A, the 17-amino
acid peptide representing the calmodulin-binding domain of smooth
muscle MLCK was compared with the amino acid sequence of the C-terminal
CaM-binding domain of gp160. The region of best fit between the two
regions is shown, with exact or near-matches in bold.
B, model of calcium-bound calmodulin ( green )
alone and ( C ) complexed with gp160 peptide
( blue ). Wild type gp160 with Ala at position 835( purple ) is shown with the helix coming out of the paper and
calmodulin wrapping around it. Note that the N terminus of calmodulin
is in the same position in both ( B and C ) and
that upon binding gp160 ( C ), the C terminus of calmodulin
moves down and the central helix region becomes a loop. The model is
based on the crystal structure of calmodulin ( 51 ) and the CaM-smooth
muscle MLCK peptide complex ( 39 ). The four Ca 2+ atoms from
the CaM-MLCK complex are shown as spheres. Ala-835 is predicted to make
a total of six van der Waals contacts with Leu-18 and Phe-19, with
distances ranging from 3.22and 3.99Å. The five hydrophobic amino
acids of calmodulin that would form unacceptable van der Waals contacts
(<2.0 Å) with the A835W mutant are colored orange (Leu-18,
Phe-19, Val-35, Leu-39, and Leu-112). These figures were prepared using
RIBBONS ( 52 ).
An alanine 835mutation to tryptophan in the gp160 peptide is predicted
to be a substitution that will maximally affect binding to calmodulin
because three-dimensional computer modeling predicts that this alanine
fits in a tight hydrophobic pocket of calmodulin. Substitution of
tryptophan for alanine is not predicted to change the overall
conformation of gp160 but is predicted to disrupt binding to
calmodulin, because the large side chain would be within 2Åof five
hydrophobic residues on calmodulin, too close to allow binding. The
ribbon structure of calmodulin (calcium saturated) is illustrated in
the absence of peptide (Fig. B ) and in its predicted
conformation when bound to wild type gp160 peptide (Fig. C ). Ala-835 of gp160 is shown in purple and the
five residues of calmodulin (Leu-18, Phe-19, Val-35, Leu-39, and
Leu-112) predicted to be within 2Åof the Trp residue of the mutant
gp160 peptide are in orange.
Calmodulin Binding by gp160 Peptides--
The effect of the A835W
mutation of gp160 on calmodulin binding was tested directly using
dansyl-calmodulin (dansyl-CaM) (Fig. ).
Dansyl-CaM is a fluorescently tagged calmodulin that binds target
proteins with high affinity and has characteristic fluorescence emission spectra when excited at 340nm, which increases upon binding
to either calcium or peptides. Dansyl-calmodulin has previously been
shown to activate calmodulin-dependent processes
normally, indicating that the labeling has no effect on the biological
activity of calmodulin ( 21 ). By titrating dansyl-CaM with increasing concentrations of peptide, dissociation constants for the
dansyl-CaMpeptide complex may be obtained ( 21 ). Peptides
corresponding to the C-terminal calmodulin-binding domain (wild type or
A835W) were tested for their ability to bind dansyl-CaM, both in the
presence and absence of calcium. Dansyl-CaM (100n M ) was
titrated with increasing concentrations of either the wild type gp160
(Fig. A ) or A835W (Fig. B ) peptides. Studies
were performed in the presence of 1m M CaCl 2,
and binding was shown to be calcium-dependent by chelating
Ca 2+ upon addition of 5m M EGTA, which
eliminated binding (data not shown). Emission spectra were obtained
using dansyl-CaM alone or dansyl-CaM plus 5µ M wild type
peptide (Fig. A ) or 5µ M A835W mutant peptide
(Fig. B ). Only the wild type peptide significantly increased the fluorescence intensity of dansyl-CaM over the wavelengths scanned. The lack of increased emission intensity with the A835W peptide indicates that it does not bind to dansyl-calmodulin. A binding
curve was generated by subtracting the fluorescence intensity of
dansyl-CaM at 485nm from the fluorescence intensity of dansyl-CaM plus
peptide for each concentration of peptide as described previously ( 21 ).
A representative curve shows saturable binding of gp160 peptide to
dansyl-CaM (Fig. C ).
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Fig. 5.
Calmodulin binding to gp160 peptide and A835W
mutant peptides of gp160. Dansyl-CaM (100n M ) was
incubated with increasing concentrations of wild type gp160 peptide,
DRVIEVVQGACRAIRHIP ( A ), or the A835W mutant peptide,
DRVIEVVQGWCRAIRHIP ( B ). After excitation at 340nm, emission
was monitored at 400-600 nm. Fluorescence spectra of dansyl-CaM alone
( empty squares ) or 5µ M peptide ( filled
squares ). The concentration-dependent binding of wild
type peptide to calmodulin ( C ) was determined by measuring
the fluorescence change at 485nm for each concentration of wild type
peptide. Data shown are representative of three independent
measurements for each peptide.
Effect of gp160A835W on FAS-mediated Apoptosis--
The effect of
the A835W mutation on gp160-enhanced FAS-mediated apoptosis was
determined by TdT staining of Jurkat cells treated with FAS antibody
for 3h. Transfection of the gp160 mutant, A835W, eliminated the
enhancement of FAS-mediated apoptosis observed with wild type gp160
(Fig. ). Apoptosis levels in
gp160A835W-transfected cells were significantly reduced from wild type
gp160-transfected cells ( p =0.009) and were nearly
identical to levels of apoptosis measured in vector-transfected
cells.
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Fig. 6.
Apoptosis in Jurkat cells transiently
transfected with wild type gp160 and the A835W point mutant of
gp160. Jurkat cells were transfected with empty vector, pSRHS, or
vector containing wild type gp160, or the mutant gp160A835W. Cells were
treated with 500ng/ml anti-FAS antibody for 3h and then
TdT-stained. Results show the mean percentage apoptotic cells±S.E. of four separate experiments. Cells transfected with gp160
underwent significantly more apoptosis in response to anti-FAS than did
either vector-transfected ( p =0.002) or
gp160A835W-transfected cells ( p =0.009).
Tet-off Jurkat Cells Expressing gp160 or gp160A835W--
To
determine more precisely the effect of the A835W mutation of gp160 on
FAS-mediated apoptosis, Jurkat Tet-off cell lines were created (as
described under "Experimental Procedures") that stably express
either wild type gp160 or gp160A835W under tetracycline control. The
tetracycline concentration-dependent expression of both
glycoproteins was measured at the mRNA level after 48-h incubation with the indicated concentrations of tetracycline (Fig.
). Representative Northern blots for
wild type gp160 and gp160A835W demonstrate an increase in mRNA
expression for both genes in response to decreasing tetracycline
concentrations (Fig. A ). Similar patterns of protein expression were also observed following removal of tetracycline and
immunoprecipitating with HIV-infected human serum indicating that both
glycoproteins were expressed at similar concentrations (data not
shown).
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Fig. 7.
Expression of gp160, gp160A835W, and
calmodulin in Tet-off Jurkat cells. Tet-off Jurkat cells
transfected with gp160 or gp160A835W were incubated for 48h in
decreasing concentrations of tetracycline (2to 0µg/ml), and
expression of gp160 or gp160A835W mRNA was measured by Northern
blot ( A ) as described under "Experimental Procedures,"
or equivalent amounts of cell lysate were separated by SDS-PAGE and
Western-blotted for calmodulin ( B ). Data are representative
Western blots from each cell line with calmodulin indicated as
CaM.
Since earlier studies had shown an increase in calmodulin levels in
cells transfected with gp160 but not a truncated form of gp160 ( 27 ), we
tested whether a single point mutation affecting only one
calmodulin-binding domain would be sufficient to eliminate increased
expression of calmodulin. Tet-off Jurkat cells transfected with gp160
or gp160A835W were incubated with decreasing concentrations of
tetracycline for 48h. Cells were washed and lysed, and equivalent amounts of protein were separated by SDS-PAGE and Western-blotted for
calmodulin (Fig. B ). Calmodulin expression inversely
correlates with tetracycline concentration in wild type gp160 cells but
not in cells expressing gp160A835W. The pattern of calmodulin
expression mirrors expression of wild type gp160 seen in Fig.
A, with an approximate 2-3-fold increase in calmodulin
expression in cells with the highest expression of gp160 (no tetracycline).
FAS-mediated Apoptosis and Caspase Activation in Tet-off gp160 and
A835W Cells--
FAS-mediated apoptosis was tested in the Tet-off cell
lines, both in the presence of tetracycline and 48h after removal
of tetracycline from the culture medium (Fig.
). In the presence of tetracycline, when
glycoprotein expression is repressed, both wild type gp160 and
gp160A835W-transfected lines respond to FAS identically, with
approximately 20-30% of total cells being apoptotic (Fig.
A ). This level of apoptosis is consistent with the effect of anti-FAS treatment on mock-transfected Jurkat cells (Fig.
A ). In cells treated with anti-FAS for 3h following
removal of tetracycline, 70% of wild type gp160-expressing cells
undergo apoptosis, an increase of 2.5-fold over uninduced cells,
whereas the gp160A835W-expressing cells show no significant increase in
apoptosis above levels seen in uninduced cells.
View larger version (30K):
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Fig. 8.
Apoptosis and caspase 3activation in Tet-off
Jurkat cells expressing gp160 or gp160A835W. A, Tet-off
Jurkat cells transfected with either gp160 or gp160A835W were treated
with anti-FAS for 3h following 48h incubation in the
presence or absence of 2µg/ml tetracycline. Apoptosis was determined
by TdT staining and manually counting 400cells/slide. B,
Tet-off Jurkat cells transfected with gp160 or gp160A835W or
untransfected Jurkat cells were incubated for 48h in the absence
of tetracycline to induce expression of glycoprotein and then treated
with 500ng/ml anti-FAS for the times indicated and lysed. Lysates were
separated by SDS-PAGE and Western-blotted for caspase 3using a
polyclonal antibody that recognizes the active subunits of caspase 3(indicated by arrow ).
Parallel effects on caspase 3activation were observed. Tet-off Jurkat
cells expressing gp160 or gp160A835W were treated with anti-FAS for the
indicated times, washed, and lysed, and equivalent protein was resolved
by SDS-PAGE and Western-blotted for caspase 3using an antibody that
recognizes the active subunits of the enzyme (Fig. B ). In
these experiments, an increase in the active subunits on Western blot
indicates activation of caspase 3.As expected, there is a substantial
increase in the amount of active subunits of caspase 3in cells
expressing wild type gp160. The presence of active caspase 3is
dramatically reduced in lysates from gp160A835W-expressing cells
compared with lysates from wild type gp160-expressing cells. Active
caspase 3can be detected as little as 1h following anti-FAS
treatment of wild type-expressing cells, whereas very little active
caspase 3is seen in the gp160A835W-expressing cells even after 4h of anti-FAS treatment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Research on HIV-1 and AIDS has recently led to development of
treatments that have significantly decreased mortality in the United
States. However, the current highly active anti-retroviral treatment
does not eliminate the virus from the body ( 28 ) and is far too
expensive and complicated to be useful in developing nations where HIV
infection is most prevalent. Therefore, understanding the interactions
between a virus and its host remains essential for developing new
therapeutic modalities. The molecular mechanisms responsible for the
decline of CD4+ lymphocytes are unknown, although there is evidence
that both FAS and FAS ligand expression are increased in AIDS patients
( 13, 29, 30 ), implicating FAS-mediated apoptosis as a potentially
important mechanism.
Earlier work has shown that expression of HIV-1 envelope glycoprotein,
gp160, overcomes a block in the FAS pathway in Molt4 cells ( 19 ), a
FAS-resistant human T-cell line deficient in hematopoietic stem cell
phosphatase. The results presented here support these earlier data and
show that the effects of gp160 are not cell line-specific. These new
data further support the hypothesis that calmodulin binding by gp160 is
biologically relevant. Truncation mutants have suggested this, both in
the work by Pan et al. ( 19 ) and by others ( 26 ) who showed
that progressive reductions in the length of the cytoplasmic tail of
gp160 had increasingly large effects on viral production in
vitro.
The long cytoplasmic tail of gp160 is a feature conserved among HIV-1,
HIV-2, and simian immunodeficiency virus. No specific function has been
attributed to the cytoplasmic region, and despite considerable
variability in the overall sequence, all known sequence variants of
HIV-1 maintain the calmodulin-binding function ( 31 ). Cells transfected
with gp160 are more sensitive to FAS-mediated apoptosis than
mock-transfected cells, and this enhancement can be blocked by
pretreatment with calmodulin antagonists (Fig. ). The enhanced
apoptosis is reduced in cells transfected with truncations of gp160
that remove most of the cytoplasmic tail of gp160, including the
C-terminal calmodulin-binding domain ( 19 ).
To determine whether the mechanism of gp160-dependent
enhanced apoptosis occurs at the level of the FAS receptor, we
investigated whether transfection of gp160 enhanced
ceramide-mediated apoptosis. Ceramide is a proposed mediator of
apoptosis produced by cleavage of sphingomyelin by a cytosolic
sphingomyelinase downstream of FADD in the FAS pathway ( 32 ). Whether
ceramide generation is necessary for FAS-mediated apoptosis remains
controversial ( 33-35 ), and we show that expression of gp160 enhances
apoptosis induced by addition of a cell-permeable, synthetic ceramide
compared with mock-transfected Jurkat cells. Calmodulin antagonists
block gp160-enhanced ceramide-mediated apoptosis, suggesting that they
function downstream of ceramide.
Caspase 3,or caspase 3-like proteases, are critical effectors that
play an important role in most types of apoptosis ( 8 ). During
activation, caspase 3is first cleaved by an upstream caspase (caspase
8in the FAS pathway) and then undergoes a second autocatalytic cleavage to form the fully active protease. Cells transfected with
gp160 and treated with FAS antibody or ceramide activate caspase 3more
rapidly than mock-transfected Jurkat cells. The data suggest that the
effects of gp160 are upstream of caspase 3activation. This supports a
previous report demonstrating that HIV infection-induced apoptosis
could be blocked by caspase inhibition ( 36 ). Similarly, TMX and TFP
block gp160-enhanced FAS- and ceramide-induced apoptosis,
suggesting a calmodulin-dependent mechanism. The molecular site of action of calmodulin antagonists in gp160-enhanced apoptosis is
currently under investigation. However, the data presented here
indicate that calmodulin binding to gp160 is an early and required step
in gp160-enhanced FAS- and ceramide-mediated apoptosis.
To confirm the effect of calmodulin binding by gp160 on FAS-mediated
apoptosis, we created a point mutation in the C-terminal CaM-binding
domain based on comparisons with the calmodulin-binding domain of MLCK
( 37-38 ). The sequence of the calmodulin-binding domains of both smooth
muscle and skeletal muscle MLCK were compared with the gp160
calmodulin-binding sequence. However, the smooth muscle MLCK was used
for computer modeling because its complex with calmodulin has been
determined by crystal structure analysis ( 39 ). An alanine to tryptophan
mutation at amino acid 835of the gp160 peptide was created that was
predicted not to change the overall amphipathic helical structure of
the region but would replace a small hydrophobic amino acid with a much
larger hydrophobic amino acid. The A835W mutant of gp160 failed to
enhance FAS-mediated apoptosis above control levels (Fig. ),
indicating that this residue is critical to gp160 function and further
supports the hypothesis that the interaction between gp160 and
calmodulin is necessary to enhance apoptosis. Peptides corresponding to
the C-terminal binding domain of the wild type gp160 and the A835W mutant were used in binding experiments with dansyl-calmodulin and
demonstrated that only the wild type gp160 peptide bound
dansyl-calmodulin. Binding of calmodulin by the wild type peptide was
Ca 2+ -dependent (data not shown), and the
affinity was identical to reported values for larger peptides that
encompassed the peptide used here ( 31 ). This is the first report
identifying a single amino acid being critical in the binding of gp160
to calmodulin, although several groups have published studies on the
effects of mutating the C terminus of gp160. Tencza et al.
( 18 ) reported that changing several basic residues to negatively
charged glutamic acid residues and single hydrophobic residues changed
to polar residues all reduced calmodulin binding and the lytic function of these peptides. Other reports have shown that gp160 binds to calmodulin and that C-terminal truncations of gp160 do not ( 40 ), but
these mutations are more likely to involve changes in tertiary structure and thus are probably less specific than the single point mutation.
Although the mechanism by which gp160 up-regulates calmodulin is not
known, the simplest explanation is that gp160 may be acting as a
calmodulin sink, binding available calmodulin, and that cells
compensate by increasing expression of calmodulin. Calmodulin
expression correlates with increased expression of wild type gp160 but
is unchanged with expression of gp160A835W, clearly suggesting that
calmodulin is a critical signaling molecule in gp160-enhanced apoptosis.
Expression of gp160 has been reported to induce apoptosis without
stimulation by FAS antibody ( 40 ). Induction of apoptosis was
dependent on calmodulin binding to gp160, as cells expressing C-terminal truncations of as few as five amino acids did not bind calmodulin and did not show increased apoptosis. This supports the
basic findings of our earlier studies ( 19 ) except that we did not
observe differences in basal apoptosis between gp160 and mock-transfected cells. There are, however, significant differences between the two studies (ranging from cell lines used to methods of
transfection and measurements of apoptosis) that make direct comparisons impossible.
There are also reports of HIV-1 and simian immunodeficiency virus
proteins other than gp160 that increase apoptosis, including Vpu ( 41 ),
Nef ( 42 ), and Tat ( 43 ). Whereas the proposed mechanisms that these
proteins use to enhance apoptosis vary, they include FAS ligand
up-regulation (Nef), caspase 8up-regulation (Tat), and all converge on
FAS-mediated apoptosis. These reports, coupled with the data presented
in this report, leave open the possibility that calmodulin plays a
central role in mediating the effects of each of these proteins.
Currently, there are no known calmodulin-dependent enzymes
directly involved in the FAS pathway, but data presented here suggest that at a minimum, calmodulin binding is required to mediate
gp160-enhanced apoptosis. It remains to be determined whether
calmodulin-dependent enzyme activity is also essential in
this process. Calmodulin-dependent processes have been
implicated in glutamate- ( 44 ), glucocorticoid- ( 45 ), tumor necrosis
factor-, and UV light ( 46 )-dependent apoptosis.
Furthermore, there are important potential sites for Ca 2+ /calmodulin action downstream of caspase 3activation,
such as death-associated protein kinase ( 47-49 ). Expression of gp160
has also been reported to increase [Ca 2+ ] i ( 50 )
which would promote calmodulin binding to target proteins and
potentially increase the activity of the
Ca 2+ /Mg 2+ -dependent endonuclease
responsible for DNA cleavage in apoptosis ( 10 ). We hypothesize that
gp160 binds to calmodulin and increases calmodulin expression,
resulting in recruitment of specific proteins to a membrane complex
that enhances the apoptotic potential of the cell. Interestingly, there
is considerable sequence homology between the death domains of many
apoptosis-inducing proteins and gp41 ( 19 ), providing an additional
potential mechanism for the recruitment of apoptotic mediating proteins
to the membrane.
In conclusion, these investigations provide strong evidence that gp160
expression enhances the cellular response to anti-FAS and increases
caspase 3activity and that these events require calmodulin binding to
gp160. The possibility that it is the increase in calmodulin expression
that is mediating the effects of gp160 on apoptosis opens a new avenue
in the study of HIV interaction with an infected cell. The use of
calmodulin antagonists and/or caspase inhibitors may provide useful
tools in future studies of viral replication and infectivity,
ultimately leading to improved therapies for AIDS and/or effective
vaccine development.
FOOTNOTES
*
This work was supported by NCI Grant CA/72823 (to
J.M.M.) from the National Institutes of Health and a Veterans
Administration Merit Review (to J.M.M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
" advertisement " in
accordance with 18U.S.C. Section
1734solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Pathology, University of Alabama at Birmingham, 70119th St. S., LHRB 509,Birmingham, AL 35294-0007.Tel.: 205-934-6666; Fax:
205-975-9927.
ABBREVIATIONS
The abbreviations used are:
HIV-1, human
immunodeficiency virus type 1;
gp, glycoprotein;
PAGE, polyacrylamide
gel electrophoresis;
TBS, Tris-buffered saline;
TdT, terminal
deoxynucleotidyltransferase;
MOPS, 4-morpholinepropanesulfonic acid;
TMX, tamoxifen;
TFP, trifluoperazine;
dansyl, 5-dimethylaminonaphthalene-1-sulfonyl;
MLCK, myosin light chain kinase;
CaM, calmodulin.
REFERENCES
TOP
ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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